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. 2022 Jan 10;13:52. doi: 10.1038/s41467-021-27692-9

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Representative images and time course plots showing the effects of PLC and PI3K inhibitors on GSDMD pore functions. Left column: no pretreatment; middle column: 10 min 10 µM U73122 (PLC inhibitor); right column: 10 min 10 µM wortmannin (PI3K inhibitor). Scale bar: 10 µm. b Whole-cell average calcium levels over many cells support the results from single cell responses. The color scheme follows green: no pretreatment; blue: pretreated with U73122; and red: pretreated with wortmannin. Line: mean value; shaded: standard deviation (SD). c Changes in single calcium flare kinetics with or without pretreatment of wortmannin, which significantly slowed-down pore dynamics by a factor of 2. Cyan lines in each panel represent the flare signals subjected to the cumulative inhibitory model fit reported. d Pixel-wise calcium fluctuation show pore dynamics changes with phosphoinositide modulation. For panel (d), statistical significance was reported from individual paired f- and t-tests with two-tailed Welch’s correction; “ref” represents the reference, ***p = 0.0003 (no-pretreat vs. wortmannin); ****p < 0.0001 (no-pretreat vs. U73122 and no-Ca²⁺).